Journal Article

PreJuSER-19855

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Improved biocytin labeling and neuronal 3D reconstruction

Marx, M.FZJ* ; Günter, R. H.FZJ* ; Hucko, W.FZJ* ; Radnikow, G.FZJ* ; Feldmeyer, D.FZJ*

2012
Nature Publishing Group Basingstoke

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Please use a persistent id in citations: doi:10.1038/nprot.2011.449

Abstract: In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

Keyword(s): Animals (MeSH) ; Brain: cytology (MeSH) ; Brain: ultrastructure (MeSH) ; Imaging, Three-Dimensional: methods (MeSH) ; Lysine: analogs & derivatives (MeSH) ; Mice (MeSH) ; Microtomy: methods (MeSH) ; Neurons: ultrastructure (MeSH) ; Osmium Tetroxide (MeSH) ; Rats (MeSH) ; Staining and Labeling: methods (MeSH) ; Osmium Tetroxide ; Lysine ; biocytin ; J

Note: This work was supported by the Deutsche Forschungsgemeinschaft (DFG; Research Group BaCoFun), the Helmholtz Association and the Helmholtz Alliance for Systems Biology. We thank A. Rollenhagen for help with the EM protocol and T. Abel for critically reading the manuscript.

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Contributing Institute(s):

1. Molekulare Organisation des Gehirns (INM-2)

Research Program(s):

1. Funktion und Dysfunktion des Nervensystems (FUEK409) (FUEK409)
2. 89571 - Connectivity and Activity (POF2-89571) (POF2-89571)

Appears in the scientific report 2012

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Database coverage:
; Current Contents - Life Sciences ; NCBI Molecular Biology Database ; Nationallizenz ; SCOPUS ; Science Citation Index Expanded ; Thomson Reuters Master Journal List ; Web of Science Core Collection

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